Dual Beneficial Effects of Methylnissolin-3-O-β-d-Glucopyranoside on Obesity-Induced Inflammatory Responses in Adipocyte-Macrophage Co-Culture

Methylnissolin-3-O-β-d-glucopyranoside (MNG) is a pterocarpan analog, which protects EA.hy926 cells against oxidative damage through the Nrf2/HO-1 pathway. However, the effects of MNG on obesity-induced inflammatory responses in adipocyte-macrophage co-culture remain unclear. A differentiated murine preadipocyte cell line (3T3-L1) was co-cultured with a murine macrophage cell line (RAW264.7). Intracellular lipid accumulation was determined using Oil Red O staining. Western blotting was performed to investigate the expression of adipogenesis- and inflammation-associated proteins. Cell culture supernatants were assayed using ELISA kits to measure the levels of proinflammatory cytokines such as interleukin 6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1). MNG inhibited lipid accumulation and the production of IL-6 and MCP-1 in the 3T3-L1 and RAW264.7 cell co-culture. Moreover, MNG inhibited the protein expression of CCAAT/enhancer-binding protein alpha (C/EBPα), C/EBPβ, peroxisome proliferator-activated receptor γ (PPARγ), cyclooxygenase 2 (COX-2), and inducible nitric oxide synthase (iNOS) under the same co-culture conditions. MNG also inhibited IL-6 and MCP-1 production compared with the co-culture control. These findings demonstrate that MNG inhibited lipid accumulation and inflammatory response by downregulating IL-6 and MCP-1 production and protein expression of C/EBPβ, C/EBPα, PPARγ, COX-2, and iNOS in co-culture conditions with 3T3-L1 and RAW264.7 cells. These results suggest that MNG may be beneficial in preventing obesity-related inflammatory status.


Introduction
Obesity is the state of low-grade systemic inflammation closely associated with an increased prevalence of metabolic diseases such as, type 2 diabetes, arteriosclerosis, cardiovascular disease, and cancer [1]. Adipose tissue can recruit and stimulate macrophages by secreting excess proinflammatory cytokines, monocyte chemotactic protein-1 (MCP-1), and free fatty acids [2]. The accumulation of macrophages in adipose tissue can result in the secretion of proinflammatory cytokines, such as interleukin 6 (IL-6), and leads to inflammatory response and obesity-induced insulin resistance in adipose tissue [3]. Therefore, reducing obesity-induced inflammation might attenuate obesity-related diseases. Red ginseng-derived saponin fraction, 6-gingerol (found in ginger), and lunasin (identified in several grains) inhibit obesity-induced inflammatory responses by inhibiting the production of inflammatory mediators MCP-1 and IL-6 in co-culture conditions with 3T3-L1 and RAW264.7 cells [4][5][6]. However, studies of the natural products used to inhibit obesityinduced inflammatory responses and their application to obesity-related inflammatory diseases remain lacking.
Methylnissolin-3-O-β-D-glucopyranoside (MNG) is a natural product of Astragalus membranaceus [7]. Astragalus membranaceus has been investigated extensively, but MNG as a single chemical entity has been rarely studied regarding its biological activities, except for Nrf2 activation, which we reported in 2020 [7] and 2021 [8]. MNG enhanced the expression of Nrf2, increased Nrf2 nuclear translocation, activated Nrf2 target proteins, and regulated cytoprotective responses to stress caused by reactive oxygen species. In addition, MNG demonstrated effects on the PI3K/Akt pathway involved in controlling Nrf2-ARE (Nrf2 antioxidant response element) activity [8]. However, the activity of MNG against obesity-induced inflammatory responses in adipocyte-macrophage co-culture has not been investigated. Therefore, the effects of MNG on obesity-induced inflammatory responses and its potential molecular mechanism in the co-culture of 3T3-L1 and RAW264.7 cells were investigated in this study.

Identification of MNG
MNG was isolated as a powder with a molecular formula of C 23 H 26 O 10 , which was determined by HRESIMS, requiring 11 degrees of unsaturation. A comprehensive analysis of the 1D and 2D NMR spectra indicated the presence of five aromatic protons, two methoxy groups, seven methines, and two methylene groups. Four spin systems, including a 1,2,3,4-tetrasubstitued benzene ring, a 1,2,4-trisubstitued benzene ring, pyranose, and CH-CH-CH2, were identified in the COSY spectrum. The spin systems were connected based on HMBC correlations, and the compound was identified as MNG ( Figure 1) [7, 8].
not been investigated. Therefore, the effects of M responses and its potential molecular mechan RAW264.7 cells were investigated in this study.

Identification of MNG
MNG was isolated as a powder with a mol determined by HRESIMS, requiring 11 degre analysis of the 1D and 2D NMR spectra indicated two methoxy groups, seven methines, and two including a 1,2,3,4-tetrasubstitued benzene rin pyranose, and CH-CH-CH2, were identified in were connected based on HMBC correlations, and (

Discussion
In this study, we investigated the effects of MNG on obesity-induced inflammatory responses in 3T3-L1 and RAW264.7 cells. MNG inhibited lipid accumulation in both 3T3-L1 and RAW264.7 cells. The inhibitory effects of 100 μM MNG and 25 μM 6-gingerol (the positive control) were similar. The compound 6-Gingerol, found in ginger, has been well reported for its anti-obesity effect [7-9]. Reportedly, 25 μM 6-gingerol inhibits lipid accumulation and obesity-induced inflammatory responses by inhibiting the production of the inflammatory mediators MCP-1 and IL-6 in co-culture conditions with 3T3-L1 and RAW264.7 cells [5]. In this study, consistent with the inhibition of intracellular lipid accumulation, MNG inhibited the production of the inflammatory mediators MCP-1 and IL-6 in co-culture conditions with 3T3-L1 and RAW264.7 cells. Studies have reported that obesity is associated with inflammation that contributes to impaired insulin signaling in adipose tissue, liver tissue, and skeletal muscle [10]. Obesity is characterized by an increase in macrophage infiltration into adipose tissue. Excessive lipid accumulation in adipocytes during obesity increases the availability of circulating fatty acids that stimulate macrophages. During the interaction between adipocytes and macrophages, excessive proinflammatory cytokines MCP-1 and IL-6 activate the inflammatory response [11]. Our results indicate that MNG inhibits obesity-induced excessive proinflammatory cytokines. In addition, MNG inhibited the protein expression of C/EBPβ, C/EBPα, PPARγ, COX-2, and iNOS in co-culture conditions with 3T3-L1 and RAW264.7 cells. In the molecular pathways related to adipogenesis and lipid accumulation, C/EBPβ, a nuclear transcription factor, activates C/EBPα and PPARγ to promote adipogenesis and lipid accumulation [12]. A study has demonstrated that an adipocyte-mediated increase in COX-2 and iNOS generates a large amount of NO, which leads to insulin resistance in adipose tissues [9,13]. Thus, our results demonstrated that MNG inhibited lipid accumulation and inflammatory response by downregulating IL-6 and MCP-1 production and protein expression of C/EBPβ, C/EBPα, PPARγ, COX-2, and iNOS in co-culture conditions with 3T3-L1 and RAW264.7 cells. These results suggest that MNG may be beneficial in preventing obesity-

Discussion
In this study, we investigated the effects of MNG on obesity-induced inflammatory responses in 3T3-L1 and RAW264.7 cells. MNG inhibited lipid accumulation in both 3T3-L1 and RAW264.7 cells. The inhibitory effects of 100 µM MNG and 25 µM 6-gingerol (the positive control) were similar. The compound 6-Gingerol, found in ginger, has been well reported for its anti-obesity effect [7][8][9]. Reportedly, 25 µM 6-gingerol inhibits lipid accumulation and obesity-induced inflammatory responses by inhibiting the production of the inflammatory mediators MCP-1 and IL-6 in co-culture conditions with 3T3-L1 and RAW264.7 cells [5]. In this study, consistent with the inhibition of intracellular lipid accumulation, MNG inhibited the production of the inflammatory mediators MCP-1 and IL-6 in co-culture conditions with 3T3-L1 and RAW264.7 cells. Studies have reported that obesity is associated with inflammation that contributes to impaired insulin signaling in adipose tissue, liver tissue, and skeletal muscle [10]. Obesity is characterized by an increase in macrophage infiltration into adipose tissue. Excessive lipid accumulation in adipocytes during obesity increases the availability of circulating fatty acids that stimulate macrophages. During the interaction between adipocytes and macrophages, excessive proinflammatory cytokines MCP-1 and IL-6 activate the inflammatory response [11]. Our results indicate that MNG inhibits obesity-induced excessive proinflammatory cytokines. In addition, MNG inhibited the protein expression of C/EBPβ, C/EBPα, PPARγ, COX-2, and iNOS in co-culture conditions with 3T3-L1 and RAW264.7 cells. In the molecular pathways related to adipogenesis and lipid accumulation, C/EBPβ, a nuclear transcription factor, activates C/EBPα and PPARγ to promote adipogenesis and lipid accumulation [12]. A study has demonstrated that an adipocyte-mediated increase in COX-2 and iNOS generates a large amount of NO, which leads to insulin resistance in adipose tissues [9,13]. Thus, our results demonstrated that MNG inhibited lipid accumulation and inflammatory response by downregulating IL-6 and MCP-1 production and protein expression of C/EBPβ, C/EBPα, PPARγ, COX-2, and iNOS in co-culture conditions with 3T3-L1 and RAW264.7 cells. These results suggest that MNG may be beneficial in preventing obesity-related inflammatory diseases. Additional reliable evidence from further research is necessary to evaluate whether MNG shows the same effects in animal models.

Cell Viability Assays
The nontoxic concentration ranges of MNG in 3T3-L1 preadipocytes and RAW264.7 cells were evaluated using an Ez-Cytox cell viability assay kit (Daeil Lab Service Co., Seoul, Korea). Cells were seeded and incubated with various concentrations of MNG for 24 h. Cell viability was measured using a microplate reader (PowerWave XS; Bio-Tek Instruments, Winooski, VT, USA) at 450 nm after reaction for 1 h at 37 • C, followed by the addition of 10% (v/v) Ez-Cytox reagent.

Adipogenic Differentiation Assays and Co-Culture
The 3T3-L1 preadipocytes were seeded and stimulated with 0.5 mM 1-isobutyl-3methylxanthine, 5 µg/mL insulin, and 1 µg/mL dexamethasone for 2 days. The culture medium was replenished with 5 µg/mL of insulin for 2 days. The culture medium was replaced every 2 days until day 8. On day 8, differentiated adipocytes were co-cultured with RAW264.7 cells.

Oil Red O Staining
Differentiated 3T3-L1 adipocytes co-cultured with RAW264.7 cells were treated with MNG, fixed with 4% paraformaldehyde solution for 1 h, and stained with Oil Red O solution for 1 h. Lipid accumulation was observed using a light microscope at a magnification of 20×. The plates were treated with 100% isopropanol, and the optical density of the eluted Oil Red O solution at 520 nm was determined using a microplate reader (PowerWave XS; Bio-Tek Instruments, Winooski, VT, USA).

Measurement of Proinflammatory Cytokine Levels
Cell culture supernatants were collected and assayed using ELISA kits specific for MCP-1 and IL-6, according to the manufacturer's protocol (R&D Systems, Minneapolis, MN, USA). The levels of MCP-1 and IL-6 were determined by measuring absorbance

Statistical Analysis
All experiments were performed in triplicate. All analyses were performed using SPSS Statistics ver. 19.0 (SPSS Inc., Chicago, IL, USA). Nonparametric comparisons of samples were conducted using the Kruskal-Wallis test to analyze the results. Differences were considered statistically significant at p < 0.05.

Conclusions
In the co-culture condition with 3T3-L1 and RAW264.7 cells, MNG inhibited lipid accumulation and production of IL-6 and MCP-1. Moreover, MNG inhibited adipogenesisand inflammation-associated proteins, including C/EBPα, C/EBPβ, PPARγ, COX-2, and iNOS, in co-culture conditions with 3T3-L1 and RAW264.7 cells. These findings provide evidence that MNG inhibits adipocyte-mediated inflammation and that MNG is a potential dietary source that has an inhibitory effect on obesity-induced inflammation.